Evaluation of Two Line Probe Assays for Rapid Detection of

Mycobacterium tuberculosis, Tuberculosis (TB) Drug Resistance, and

Non-TB Mycobacteria in HIV-Infected Individuals with Suspected TB
Anne F. Luetkemeyer,a Michelle A. Kendall,b Xingye Wu,b Maria Cristina Lourenço,c Ute Jentsch,d Susan Swindells,e Sarojini S. Qasba,f
Jorge Sanchez,g Diane V. Havlir,a Beatriz Grinsztejn,c Ian M. Sanne,h,i Cynthia Firnhaber,h,i Adult AIDS Clinical Trials Group A5255
Study Team
HIV/AIDS Division, San Francisco General Hospital, University of California, San Francisco, California, USAa; Center for Biostatistics in AIDS Research, Harvard School of
Public Health, Boston, Massachusetts, USAb; Instituto de Pesquisa Clinica Evandro Chagas, Fundacao Oswaldo Cruz, Rio de Janeiro, Brazilc; Clinical Laboratory Services and
School of Pathology, University of the Witwatersrand, Johannesburg, South Africad; Division of Infectious Diseases, University of Nebraska Medical Center, Omaha,
Nebraska, USAe; Montgomery County Health Department, Silver Spring, Maryland, USAf; Asociación Civil Impacta Salud y Educacioó, Lima, Perug; Clinical HIV Research
Unit, Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, South Africah; Right to Care, Johannesburg, South Africai

Limited performance data from line probe assays (LPAs), nucleic acid tests used for the rapid diagnosis of tuberculosis (TB),
nontuberculosis mycobacteria (NTM), and Mycobacterium tuberculosis drug resistance are available for HIV-infected individu-
als, in whom paucibacillary TB is common. In this study, the strategy of testing sputum with GenoType MTBDRplus (MTBDR-
Plus) and GenoType Direct LPA (Direct LPA) was compared to a gold standard of one mycobacterial growth indicator tube
(MGIT) liquid culture. HIV-positive (HIVⴙ) individuals with suspected TB from southern Africa and South America with <7
days of TB treatment had 1 sputum specimen tested with Direct LPA, MTBDR-Plus LPA, smear microscopy, MGIT, biochemical
identification of mycobacterial species, and culture-based drug-susceptibility testing (DST). Of 639 participants, 59.3% were
MGIT M. tuberculosis culture positive, of which 276 (72.8%) were acid-fast bacillus (AFB) smear positive. MTBDR-Plus had a
sensitivity of 81.0% and a specificity of 100%, with sensitivities of 44.1% in AFB smear-negative versus 94.6% in AFB smear-posi-
tive specimens. For specimens that were positive for M. tuberculosis by MTBDR-Plus, the sensitivity and specificity for rifampin
resistance were 91.7% and 96.6%, respectively, and for isoniazid (INH) they were 70.6% and 99.1%. The Direct LPA had a sensi-
tivity of 88.4% and a specificity of 94.6% for M. tuberculosis detection, with a sensitivity of 72.5% in smear-negative specimens.
Ten of 639 MGIT cultures grew Mycobacterium avium complex or Mycobacterium kansasii, half of which were detected by Di-
rect LPA. Both LPA assays performed well in specimens from HIV-infected individuals, including in AFB smear-negative speci-
mens, with 72.5% sensitivity for M. tuberculosis identification with the Direct LPA and 44.1% sensitivity with MTBDR-Plus.
LPAs have a continued role for use in settings where rapid identification of INH resistance and clinically relevant NTM are
priorities.

R apid laboratory identification of tuberculosis (TB) and Myco-

bacterium tuberculosis drug resistance are critical to ensure
timely initiation of therapy, to inform appropriate TB therapy,
and to facilitate infection control. Early diagnosis of TB and drug-
resistant disease is of particular importance in human immuno-
deficiency virus (HIV)-infected individuals, as delay of therapy (1,
2) and drug-resistant TB (3) can be devastating in those with com-
promised immune systems. Diagnosis of TB in HIV can be a par-
ticular challenge, as 24% to 61% of HIV coinfected individuals
with pulmonary TB have acid-fast bacillus (AFB)-smear-negative
sputum (2). Culture-based testing for M. tuberculosis and M. tu-
berculosis drug resistance requires an unacceptably long turn-
around for results, is limited by contamination rates, and requires
considerable infrastructure and human resources.
Molecular line probe assays (LPA) permit rapid diagnosis of
TB, isoniazid and rifampin resistance, and clinically relevant
non-M. tuberculosis mycobacteria. In these assays, DNA or RNA is
isolated from culture or direct (i.e., sputum) respiratory samples
and then amplified and reverse hybridized onto a nitrocellulose
strip with immobilized probes for different mycobacteria or for
mutations that confer resistance. These strips can be quickly in-
terpreted using a template, with the entire testing process taking a
day or less in most cases. In 2008, the World Health Organization
endorsed the use of line probe assays for detection of M. tubercu-
losis drug resistance; however, use was recommended on culture
specimens and AFB-positive (AFB⫹) sputum specimens only,
given the lack of data for use in AFB-negative sputum (4). Given
the prevalence of paucibacillary disease in HIV-TB coinfection, it
is important to understand line probe performance across the
spectrum of bacillary loads in HIV-infected persons, in whom
there are limited data to inform use of line probe assays.
The GenoType MTBDRplus (MTBDR-Plus) (Hain Lifesciences
GmBH, Nehren, Germany) identifies rifampin (RIF) and isonia-
zid (INH) resistance by detecting the most common mutations of
the rpoB gene and the katG and inhA genes, respectively, and can
be used on both cultured and direct specimens. MTBDR-Plus LPA
Received 3 October 2013 Returned for modification 9 November 2013
Accepted 8 January 2014
Published ahead of print 15 January 2014
Editor: K. C. Carroll
Address correspondence to Anne F. Luetkemeyer, aluetkemeyer@php.ucsf.edu.
Copyright © 2014, American Society for Microbiology. All Rights Reserved.
doi:10.1128/JCM.02639-13

1052 jcm.asm.org Journal of Clinical Microbiology p. 1052–1059 April 2014 Volume 52 Number 4

RIF and INH resistance results can be interpreted only if M. tuber-
culosis has been successfully identified by the assay probe for it.
Several studies have suggested excellent performance of MTBDR-
Plus in AFB-negative direct specimens with interpretable results
in 80.0% to 95.8% of specimens (5, 6), but interpretable test rates
have been as low as 13.7% in one series (7). Of note, these studies
were conducted in populations that were either HIV uninfected or
HIV status unknown. The GenoType mycobacteria direct line
probe assay (Direct LPA) (also Hain Lifesciences GmbH) is an-
other line probe assay that uses nucleic acid sequence-based am-
plification (NASBA) to identify the mycobacterial species-specific
23S rRNA, thus differentiating the M. tuberculosis complex from
four other clinically relevant mycobacterial species, Mycobacte-
rium avium, M. intracellulare, M. kansasii, and M. malmoense. The
nontuberculosis species M. avium and M. intracellulare (collec-
tively known as the M. avium complex [MAC]) and M. kansasii
are important causes of pulmonary and disseminated disease in
HIV-infected patients (8–11). Importantly, these pathogens
would be missed with rapid diagnostics that identify only Myco-
bacterium tuberculosis. The Direct LPA is designed for use on di-
rect sputum specimens. Data are limited for AFB-negative direct
sputum samples and in HIV-infected populations, but small stud-
ies have reported sensitivities of 69.0% to 89.6% (6, 12). We
sought to determine the accuracy of the MTBDR-Plus and Direct
LPA in HIV-infected TB subjects, with both AFB⫹ and AFB-neg-
ative sputum specimens, in comparison to liquid culture. We also
evaluated the incremental yield of a second Direct LPA performed
on an additional sputum specimen as well as an additional
MTBDR-Plus LPA performed on mycobacterial culture.
MATERIALS AND METHODS
Study population. This was a cross-sectional analysis of HIV-infected
individuals with suspected tuberculosis enrolled from 7 sites: Rio de Ja-
neiro, Brazil; Lima, Peru; 2 sites in Botswana (Gaborone and Molepole);
and 3 sites in South Africa (1 in Johannesburg and 2 in Durban). Quali-
fying participants on ⬍7 days of TB therapy were referred from clinical
care to the research sites with either (i) confirmed TB (sputum AFB⫹ or
culture positive) or (ii) probable TB (no smear- or culture-positive spec-
imen but empirical TB treatment anticipated based on clinical symp-
toms). Clinical TB symptoms and signs were defined as one or more of the
following: fever for ⬎2 weeks, unintentional weight loss, night sweats,
radiographic findings compatible with TB, or contact with TB-infected
individuals. All participants provided one sputum specimen, with a min-
imum volume of 3 ml, either expectorated or induced, tested for the
following: AFB smear, liquid mycobacterial culture, GenoType Direct
LPA (decontaminated sediment), and GenoType MTBDR-Plus (decon-
taminated sediment and culture). The first 109 participants enrolled pro-
vided a second sputum specimen which was tested with AFB smear as well
as Direct LPA and MTBDR-Plus LPA on decontaminated sediment. All
participants with initial negative M. tuberculosis culture or with contam-
inated/missing results were evaluated 24 weeks after enrollment for all
interim mycobacterial results and follow-up TB status. Follow-up TB⫹
cases were defined as those with sputum cultures positive for TB after
enrollment. Lab testing occurred at one of three laboratories: (i) Contract
Laboratory Services in Johannesburg, South Africa, (ii) Fundação Os-
waldo Cruz Instituto de Pesquisa Clinica Evandro Chagas Laboratório de
Bacteriologia, Rio de Janeiro, Brazil, and (iii) Blufstein Laboratório
Clínico, Lima, Peru.
Conventional microbiology testing. Sputum samples were digested
and decontaminated with 1% N-acetyl-L-cysteine–sodium hydroxide
(NALC/NaOH) as outlined by Kent and Kubica (13). After digestion and
decontamination, specimens were neutralized and centrifuged at 3,000 ⫻
g for 15 min. AFB smears were prepared from decontaminated sediment
April 2014 Volume 52 Number 4
TB Line Probe Assays in HIV-Positive Patients
with auramine and a potassium permanganate counterstain and evalu-
ated with a Lumin portable light-emitting diode (LED) objective (40⫻)
(LW Scientific, Lawrenceville, GA) for a minimum of 100 fields, using the
WHO/International Union against Tuberculosis and Lung Disease
(IUALTD) scale for fluorescence microscopy (FM) (14). Sediment was
prepared for culture in mycobacterial growth indicator tube (MGIT) cul-
ture (Becton, Dickinson) according to the manufacturer’s instructions
(15). In cultures with mycobacterial growth, identification to the species
level was performed using biochemical testing (13). Cultures in which the
species was identified as M. tuberculosis underwent drug susceptibility
testing using Bactec MGIT streptomycin, INH, RMP, and ethambutol
(SIRE) (16), and the critical concentration was 1.0 ␮g/ml for RIF and 0.1
␮g/ml for INH. Mycobacterial blood cultures were performed at clinician
discretion using Bactec 9050 or Myco/F Lytic (both Becton, Dickinson).
Line probe assay testing. Testing was conducted in accordance with
manufacturer’s recommendations and was performed by laboratory staff
members trained by the manufacturer. DNA/RNA extraction, PCR, and
reverse hybridization were conducted in separate rooms to minimize con-
tamination. Tests were performed and interpreted without knowledge of
culture results.
GenoType MTBDRplus (version 1.0). Testing was performed on de-
contaminated sputum sediment as well as culture with confirmed TB
growth. Briefly, 500 ␮l of decontaminated sediment was centrifuged and
resuspended with 100 ␮l ultrapure water or 1,000 ␮l of bacterial suspen-
sion heat killed at 95°C for 20 min. DNA was extracted using an ultrasonic
bath. Multiplex PCR was performed using HotStar Taq DNA polymerase
250 U (Qiagen GmBH, Hilden, Germany), with 40 amplification cycles
for sediment and 30 cycles for cultured specimens. Hybridization was
performed with a TwinCubator (Hain Lifescience GmbH). Test strips
were interpreted in a two-step procedure. (i) The presence or absence of
M. tuberculosis was determined and (ii) for strips with M. tuberculosis
present, INH and RIF susceptibilities were interpreted. Strips that could
not be definitively interpreted were repeated if possible using extracted
DNA; definitive test results were reported from the second test or the final
test results was reported as “indeterminate.” MTBDR-Plus drug suscep-
tibility results were interpreted only from specimens in which M. tuber-
culosis was present. Strips with mixed resistance results (i.e., both wild-
type and resistance mutations present) were reported as resistant for
sensitivity and specificity calculations.
GenoType direct line probe (version 4.0). RNA was extracted from a
500-␮l resuspended decontaminated pellet using a magnet separator
(Hain Lifescience GmbH), followed by denaturation and isothermal nu-
cleic acid amplification performed on 10 ␮l purified RNA. We used 20 ␮l
of amplified solution hybridization with a TwinCubator (Hain Lifescience
GmbH). Test strips were interpreted as follows. First, the presence of the
conjugate control band was confirmed, and then species identification
was interpreted according to a manufacturer-provided template. Strips
lacking conjugate control or lacking amplification control (in the absence
of positive banding indicating species) were considered uninterpretable
and the specimen was rerun if possible. As with MTBDR-Plus LPA, if
repeat testing provided a definitive result, this was recorded; otherwise,
final results were recorded as “indeterminate.”
Statistical methods. Sensitivity and specificity were calculated using
standard epidemiological methods. The gold standard comparator for M.
tuberculosis detection was liquid culture (MGIT), for mycobacterial iden-
tification it was biochemical species determination, and for INH and RIF
drug susceptibility it was MGIT SIRE testing. Because of indeterminate
results, sensitivity and specificity were calculated both from an intent-to-
screen approach, where indeterminate LPA test results were interpreted as
false negatives, and from the subgroup for which all test results for LPA
and M. tuberculosis culture were definitive. In the participants with two
specimens and among those, the participants who had a negative result on
the first specimen, the incremental yield was calculated as the number of
second specimens that were positive divided by the number of first spec-
imens that were negative. The 95% confidence intervals (CIs) on these
jcm.asm.org 1053
Luetkemeyer et al.

TABLE 1 Baseline characteristics

Median (quartile 1, quartile 3) or n (%) for:
Data by result of MGIT culture
Participant characteristics Total (n ⫽ 639) Positive (n ⫽ 379) Negative (n ⫽ 224) Failure/missing (n ⫽ 36) Pa
Age (yr) 36 (30, 42) 35 (29, 41) 38 (33, 45) 36 (31, 42.5) ⬍0.001
Male 358 (56.0) 202 (53.3) 129 (57.6) 27 (75.0) 0.306

Site of enrollment
Southern Africa 438 (68.5) 316 (83.4) 101 (45.1) 21 (58.3) ⬍0.001
South America 201 (31.5) 63 (16.6) 123 (54.9) 15 (41.7)

CD4 count at enrollmentb

CD4 ⱕ 50 cells/mm3 135 (21.3) 69 (18.4) 52 (23.5) 14 (38.9) 0.129
CD4 ⱕ 350 cells/mm3 503 (79.5) 316 (84.0) 156 (70.6) 31 (86.1) ⬍0.001
BMI (kg/m2)c 20.8 (18.3, 23.4) 20.8 (18.3, 23.1) 21.1 (18.3, 23.7) 20.1 (17.9, 22.2) 0.573
Abnormal CXRd 481 (76.6) 296 (79.6) 157 (71.0) 28 (80.0) 0.018

TB symptoms reportede
Coughf 621 (97.6) 373 (98.9) 215 (96.0) 33 (94.3) 0.016
Feverg 399 (63.0) 222 (59.5) 155 (69.2) 22 (61.1) 0.018
Night sweatsh 450 (70.8) 274 (72.7) 151 (67.7) 25 (69.4) 0.196
Unintentional weight lossi 567 (90.3) 347 (93.8) 189 (84.8) 31 (88.6) ⬍0.001
Wastingj 350 (55.1) 213 (56.5) 120 (53.8) 17 (48.6) 0.522

On ART at time of sputum collection 143 (22.4) 71 (18.7) 63 (28.1) 9 (25.0) 0.007
On TB treatment (⬍7 days) at time of 152 (23.8) 109 (28.8) 32 (14.3) 11 (30.6) ⬍0.001
sputum collection
AFB FM smear positivityk 301 (47.3) 276 (72.8) 14 (6.3) 11 (32.4) ⬍0.001
a
P values were for comparisons of MGIT culture positive versus negative. The Wilcoxon test was used for age and body mass index (BMI), and the chi-square test was used for
others.
b
Due to missing data, sample sizes were 633, 376, 221, and 36, respectively.
c
Due to missing data, sample sizes were 631, 375, 221, and 35, respectively.
d
Due to missing data, sample sizes were 628, 372, 221, and 35, respectively.
e
Reported in past 30 days, any duration.
f
Due to missing data, sample sizes were 636, 377, 224, and 35, respectively.
g
Due to missing data, sample sizes were 633, 373, 224, and 36, respectively.
h
Due to missing data, sample sizes were 636, 377, 223, and 36, respectively.
i
Due to missing data, sample sizes were 628, 370, 223, and 35, respectively.
j
Due to missing data, sample sizes were 635, 377, 223, and 35, respectively.
k
Due to missing data, sample sizes were 637, 379, 224, and 34, respectively.

measures were calculated using Wilson’s score binomial method. Two-
sided Fisher’s exact and chi-square tests were used to compare sensitivities
and specificities within subgroups, with results considered significant
when alpha was ⬍0.05; other statistical comparisons were done using
two-sided Wilcoxon and chi-square tests.
The study was reviewed and approved by ethics committees at all
participating sites and all participants gave written informed consent.
RESULTS
From September 2009 to October 2011, 639 eligible study partic-
ipants with suspected TB provided sputum for evaluation. The
median age was 36 years (interquartile range [IQR], 30, 42), 56.0%
were male, and 68.5% were enrolled from Southern Africa (South
Africa and Botswana) and 31.5% from South America (Brazil and
Peru). The median CD4⫹ cell count was 151 cells/mm3 (IQR, 61,
308) with 22.4% receiving antiretroviral treatment (ART) and
23.8% having initiated TB treatment at the time of sputum collec-
tion (median 3 days [IQR, 1, 5]). Table 1 shows baseline charac-
teristics and predictors of M. tuberculosis MGIT positivity.
Figure 1 indicates the specimen flow. There were 639 speci-
mens that yielded smear microscopy, MGIT culture, Direct LPA,
and MTBDR LPA results. MGIT was positive for M. tuberculosis in
379/639 (59.3%), of which 276 (72.8%) were AFB smear positive.
MGIT was positive for nontuberculosis mycobacterium (NTM)
in 14/639 (2.2%) (9 MAC, 1 M. kansasii, and 4 others); no MGIT
culture yielded both TB and a second mycobacterial species. Of
MGIT cultures, 210/639 (32.9%) were negative for any growth,
27/639 (4.2%) were contaminated, and 9/639 (1.4%) had no re-
sults due to site/lab error. Follow-up evaluation of participants
with MGIT TB-negative or contaminated/missing results identi-
fied an additional 7 (1.1%) pulmonary M. tuberculosis culture-
positive cases.
MTBDR-Plus LPA. MTBDR LPA was positive for M. tubercu-
losis in 315/639 (49.3%) specimens, negative for mycobacteria in
314 (49.1%), and had no available result in 10 (1.6%) due to test
failure (7) or site/lab error (3). Table 2 provides the MTBDR LPA
and MGIT results for the detection of M. tuberculosis in sputum,
with MGIT as the gold standard. With the use of an intent-to-
screen approach (Table 3), MTBDR correctly identified M. tuber-
culosis in 306 of 378 M. tuberculosis culture-positive specimens
(sensitivity, 81.0% [CI, 76.7%, 84.6%]) with a specificity of 100%
(224/224 [CI, 98.3%, 100%]). When evaluating the 595 specimens
with interpretable results for both MGIT and MTBDR LPA, we

1054 jcm.asm.org Journal of Clinical Microbiology

 TB Line Probe Assays in HIV-Positive Patients

FIG 1 Specimen flow schematic.

found that sensitivity was 82.3% (306/372 [CI, 78.1%, 85.8%])
and specificity was 100% (223/223 [CI, 98.3%, 100%]). MTBDR
M. tuberculosis detection varied by AFB smear status, with sensi-
tivity of 95.6% (261/273 [CI, 92.5%, 97.5%]) in smear-positive
versus 45.5% (45/99 [CI, 36.0%, 55.2%]) smear-negative speci-
mens (Fisher’s exact test, P ⬍ 0.001) (Table 3).
Of the 379 MGIT TB-positive specimens, gold standard con-
ventional RIF and INH DST testing by MGIT was successfully
performed on 374 (98.7%); an additional two specimens had only
RIF DST results. Of these, 5/376 (1.3%) were RIF monoresistant,
15/374 (4.0%) were INH monoresistant, and 19/374 (5.1%) were
both RIF and INH resistant (MDR) (Table 4).
In an intent-to-screen analysis, MTBDR performed on spu-
tum identified 22/24 RIF-resistant specimens (sensitivity,
91.7% [CI, 74.2%, 97.7%]) with specificity of 96.6% (339/351
[CI, 94.1%, 98.0%]). When restricted to the 282 samples with
interpretable results for both MTBDR and MGIT DST, sensi-
tivity was 100% (22/22 [CI, 85.1%, 100%]) for RIF resistance
and specificity was 95.4% (248/260 [CI, 92.1%, 97.3%]).
Within specimens with interpretable results, sensitivity was not
affected by smear status; results were smear positive 100%
(20/20 [CI, 83.9%, 100%]) versus smear negative 100% (2/2
[CI, 34.2%, 100%]) (Fig. 2). In 66 MGIT M. tuberculosis-posi-
tive specimens with interpretable DST results, MTBDR LPA did
not detect M. tuberculosis and thus yielded no LPA DST test result;
of these, 65 were RIF susceptible and 1 was RIF resistant. MTBDR

TABLE 2 Mycobacterial detection by MGIT versus line probe assays

MGIT results (n [%])
M. tuberculosis NTM MAC or Other NTM No mycobacterial Not
Sample type present only M. kansasii only Contaminated growth available Total
MTBDR-plus LPA results
M. tuberculosis present 306 (80.7) 0 6 (22.2) 0 3 (33.3) 315
No M. tuberculosis present 66 (17.4) 14 (100.0) 21 (77.8) 209 (99.5) 4 (44.4) 314
Not available 7 (1.8) 0 0 1 (0.5) 2 (22.2) 10
Total 379 (100.0) 14 (100.0) 27 (100.0) 210 (100.0) 9 (100.0) 639

Direct LPA results 0

M. tuberculosis present 334a (88.1) 0 6 (22.2) 12 (5.7) 3 (33.3) 355
MAC or M. kansasii 0 5 (50.0) 1 (25.0) 0 1 (0.5) 0 7
Indeterminate 3 (0.8) 0 0 0 2 (1.0) 2 (22.2) 7
No mycobacteria present 36 (9.5) 5 (50.0) 3 (75.0) 21 (77.8) 194 (92.4) 2 (22.2) 261
Not available 6 (1.6) 0 0 0 1 (0.5) 2 (22.2) 9
Total 379 (100.0) 10 (100.0) 4 (100.0) 27 (100.0) 210 (100.0) 9 (100.0) 639
a
M. tuberculosis and MAC were detected in 3 out of the 334 specimens by Direct LPA.

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Luetkemeyer et al.

TABLE 4 Detection of rifampin and isoniazid drug resistance by

209/221 (94.6) (90.8, 96.9)

212/224 (94.6) (90.9, 96.9)

223/223 (100) (98.3, 100)

224/224 (100) (98.3, 100)

culture-based drug susceptibility testing versus MTBDR line probe assay
Culture-based DST results (n [%])

M.
tuberculosis Not
a
Drug and result Resistant Susceptible negative available Total
Rifampin MTBDR-Plus
Detection specificity (no. positive/no. detected [%]) (95% CI)

LPA results

NAc

NAc
All

Resistant 22 (91.7) 12 (3.4) 0 2 (5.1) 36

Susceptible 0 248 (70.5) 0 10 (25.6) 258
Indeterminate 1 (4.2) 20 (5.7) 0 0 21
195/207 (94.2) (90.1, 96.7)

198/210 (94.3) (90.3, 96.7)

210/210 (100) (98.2, 100)

210/210 (100) (98.2, 100)

M. tuberculosis negative 1 (4.2) 65 (18.5) 223 (99.6) 25 (64.1) 314
Not available 0 7 (2.0) 1 (0.4) 2 (5.1) 10
Total 24 (100.0) 352 (100.0) 224 (100.0) 39 (100.0) 639

Isoniazid MTBDR-plus
LPA results
Resistant 24 (70.6) 3 (0.9) 0 2 (4.8) 29
Susceptible 4 (11.8) 257 (75.8) 0 12 (28.6) 273
AFB⫺

Indeterminate 1 (2.9) 12 (3.5) 0 0 13

NAc

NAc

M. tuberculosis negative 5 (14.7) 60 (17.7) 223 (99.6) 26 (61.9) 314

Not available 0 7 (2.1) 1 (0.4) 2 (4.8) 10
Total 34 (100.0) 339 (100.0) 224 (100.0) 42 (100.0) 639
14/14 (100) (78.5, 100)
13/13 (100) (77.2, 100)

14/14 (100) (78.5, 100)

14/14 (100) (78.5, 100)

a
Nineteen specimens were RIF and INH resistant (MDR TB).

mixed RIF resistance was present in 2 specimens, 1 RIF resistant

AFB⫹

and 1 RIF sensitive by MGIT DST.

NAc

NAc

Overall, MTBDR detected 24/34 INH-resistant specimens

(sensitivity, 70.6% [CI, 53.8%, 83.2%]) as identified by conven-
374/378 ⫽ 98.9) (97.3, 99.6)

tional DST, with specificity of 99.1% (335/338 [CI, 97.4%,

334/370 (90.3) (86.8, 92.9)
306/372 (82.3) (78.1, 85.8)
374/378 (98.9) (97.3, 99.6)

334/378 (88.4) (84.7, 91.2)

306/378 (81.0) (76.7, 84.6)

99.7%]). When restricted to specimens with both MTBDR and

conventional DST results, sensitivity for INH resistance was
All available LPA test results were used for the calculations; indeterminate was considered false negative in the calculations.

85.7% (24/28 [CI, 68.5%, 94.3%]) and specificity was 98.8% (257/
260 [CI, 96.7%, 99.6%]). Sensitivity for INH resistance was not
impacted by smear status; results were smear positive 84.6%
(22/26 [CI, 66.5%, 93.8%]) versus smear negative 100% (2/2 [CI,
Specificity was not calculated, as LPA tests were performed on known M. tuberculosis-positive cultures only.
b
TABLE 3 Sensitivities and specificities of line probe assays for M. tuberculosis detectiona

All

34.2%, 100%]); however, there were only 2 INH-resistant smear-

Detection sensitivity (no. positive/no. detected [%]) (95% CI)

negative specimens (Fig. 2). MTBDR LPA yielded mixed INH re-
100/103 (97.1) (91.8, 99.0)

100/103 (97.1) (91.8, 99.0)

sistance results for 3 specimens, 2 INH resistant and 1 INH sensi-

74/102 (72.5) (63.2, 80.3)
45/102 (44.1) (34.9, 53.8)
74/99 (74.7) (65.4, 82.3)
45/99 (45.5) (36.0, 55.2)

tive by MGIT DST.

Of the 19 specimens identified as MDR TB by MGIT DST,
MDRTB LPA correctly identified 16/19 (84.2%), did not yield
interpretable results in 1 (5.3%), did not detect M. tuberculosis and
⫺
AFB

260/271 (95.9) (92.9, 97.7)

261/273 (95.6) (92.5, 97.5)
274/275 (99.6) (98.0, 99.9)

260/276 (94.2) (90.8, 96.4)

261/276 (94.6) (91.2, 96.7)
274/275 (99.6) (98.0, 99.9)

One LPA test result was not available due to site error.
⫹
AFB

MTBDR-Plus (sputum)

MTBDR-Plus (sputum)
both MGIT and LPA

MTBDR-Plus (culture)

MTBDR-Plus (culture)
Intent-to-screen resultsb
Interpretable results for

Direct line probe

Direct line probe

Result type

FIG 2 MTBDR-Plus LPA by smear status.

a

1056 jcm.asm.org Journal of Clinical Microbiology


FIG 3 Direct LPA by smear status.
thus yielded no test result in 1 (5.3%), and was not MDR in 1
(5.3%; MDRTB LPA identified RIF resistance only).
Yield of second MTBDR LPA and MTBDR LPA on culture.
Performing MTBDR LPA on a second sputum sample identified 3
more MGIT M. tuberculosis-positive cases out of 109 participants
with two sputa tested and 81 of these with the first tested as M.
tuberculosis negative, for an incremental yield of 3.7% (CI, 1.3%,
10.3%) and identified no additional drug resistance cases. Testing
MGIT M. tuberculosis-positive culture specimens with MTBDR
LPA did not improve the yields of drug resistance, detecting 22/24
(91.7%) of RIF-resistant and 28/34 (82.4%) INH-resistant speci-
mens compared to conventional DST.
Direct LPA. The Direct LPA was positive for M. tuberculosis in
355/639 (55.6%) specimens tested, positive for M. kansasii or M.
avium only in 9 (1.4%), negative for mycobacteria in 261 (40.8%),
indeterminate in 7 (1.1%), and with no available result in 9
(1.4%), due to test failure (6) or site/lab error (3). Of the 7 Direct
LPA indeterminate results, 3 were AFB smear positive and 4 were
AFB smear negative. Direct LPA was positive for NTM in 10/639
(4 MAC, 3 M. kansasii, and 3 with mixed infections with both M.
tuberculosis and MAC detected) (Table 2). Five of the 10 MAC or
M. kansasii cases detected by MGIT culture were detected by Di-
rect LPA.
Direct LPA correctly identified 334/378 (sensitivity, 88.4% [CI,
84.7%, 91.2%]) of MGIT TB positive specimens (Table 3), iden-
tifying an additional 9 specimens with M. tuberculosis in which
MGIT failed, due to contamination (6) and site/lab error (3) (Ta-
ble 2). Of the 591 with interpretable MGIT and Direct LPA results,
the sensitivity of Direct LPA for M. tuberculosis detection by AFB
smear grade was as follows (Fig. 3): smear negative 74.7% (74/99
[CI, 65.4%, 82.3%]), scanty 66.7% (10/15 [CI, 41.7%, 84.8%]),
1 ⫹ 97.1% (67/69 [CI, 90.0%, 99.2%]), 2 ⫹ 95.9% (47/49 [CI,
86.3%, 98.9%]), 3 ⫹ 98.6% (136/138 [CI, 94.9%, 99.6%]), with an
overall sensitivity in smear-positive of 95.9% (260/271 [CI, 92.9%,
97.7%]) and in smear-negative specimens of 74.7% (74/99 [CI,
65.4%, 82.3%]). Of the 221 MGIT M. tuberculosis-negative spec-
imens, Direct LPA was negative in 209 (specificity 94.6% [CI,
90.8%, 96.9%]), with slightly lower specificity in smear-negative
(195/207 or 94.2% [CI, 90.1%, 96.7%]) than smear-positive spec-
April 2014 Volume 52 Number 4
TB Line Probe Assays in HIV-Positive Patients
imens (14/14 or 100% [CI, 78.5%, 100%; Fisher’s exact test P ⫽
1.0 for the comparison). Direct LPA sensitivity did not differ sig-
nificantly (chi-square test P ⫽ 0.46) in those with CD4⫹ values of
ⱕ50 versus ⬎50 cells/mm3 (59/67 or 88.1% versus 273/300 or
91.0%, respectively). Of the 12 MGIT TB-negative and Direct LPA
M. tuberculosis-positive specimens, 9 were assessed as clinical TB
(no positive culture reported but responded to empirical TB treat-
ment) and 3 as not TB in follow-up evaluations at 24 weeks.
Incremental yield of second Direct LPA. In the subset of 109
participants with two sputa tested and the first tested as M. tuber-
culosis-negative in 70, the second sputum with Direct LPA identi-
fied 1 additional MGIT TB⫹ case (incremental yield 1.4% [CI,
0.3%, 7.7%]).
DISCUSSION
This study demonstrates that MTBDR and direct line probe assays
can be used effectively in HIV-infected patients undergoing TB
evaluation, including those with AFB smear-negative specimens.
For identification of M. tuberculosis drug resistance, MTBDR
demonstrated similar performance in an HIV-infected popula-
tion as has been previously shown in HIV-uninfected populations
(17), detecting 91.7% of rifampin resistance and 70.6% of INH
resistance. Sensitivities increased to 100% for RIF resistance and
85.7% for INH resistance when assays were restricted to samples
with results interpretable by both MGIT and LPA.
When considering performance by AFB smear status, the Di-
rect and MTBDR line probes both had excellent sensitivity of
95.6% to 95.9% on AFB⫹ direct sputum samples. In AFB-negative
specimens, the Direct line probe identified culture-confirmed M.
tuberculosis in 74.7% of AFB-negative and 66.7% of scanty smear-
positive specimens, similar to the sensitivity of 68 to 75% (18)
reported with Xpert MTB/RIF in AFB-negative specimens (19,
20). MTBDR was less sensitive than the Direct line probe for M.
tuberculosis detection, but did identify culture-confirmed M. tu-
berculosis in nearly half (45.5%); these specimens were able to
undergo rapid drug susceptibility testing as well. An updated ver-
sion 2.0 of MTBDR-Plus was released in 2011 with reported im-
proved sensitivity in AFB-negative specimens, ranging from 58%
to 80% (21, 22), further bolstering the ability to use this assay in
smear-negative specimens.
The landscape of M. tuberculosis diagnostics has undergone
remarkable changes with the development and accelerated rollout
of rapid, self-contained PCR platforms such as the Xpert MTB/
RIF. The Xpert MTB/RIF provides an attractive alternative to line
probe assays, which requires skilled laboratory staff, specialized
equipment, and dedicated PCR space to reduce the risk of con-
tamination. Given the advantages of this increasingly available
technology, what is the current role of line probe assays for rapid
detection of M. tuberculosis, NTM, and M. tuberculosis drug resis-
tance? Line probe assays are still important tools for both current
TB clinical care and research due to several advantageous aspects.
First, the MTBDR assay is one of the few rapid diagnostics that
evaluates INH susceptibility, which is not part of the current
Xpert MTB/RIF test. For clinical trials of new TB treatment
regimens, establishing INH susceptibility is an enrollment cri-
terion for many drug-sensitive protocols (e.g., Evaluation of early
bactericidal activity in pulmonary tuberculosis with clofazimine
(C)-TMC207 (J)-PA-824 (Pa)-pyrazinamide (Z) (NC-003) [http
://clinicaltrials.gov/ct2/show/NCT01691534?term⫽NC003&ran
k⫽1]) and MDR-TB protocols (23). Rapid identification with line
jcm.asm.org 1057
Luetkemeyer et al.
probes can facilitate trial conduction and help ensure inclusion of
appropriate participants. In clinical care, there has been debate
about the significance of INH monoresistance and effects on TB
treatment outcomes (24–27). However, a South African study
(27) and a meta-analysis (26) have both demonstrated poor TB
outcomes with INH monoresistance, and two studies suggest that
early detection of INH resistance with modification of treatment
led to better outcomes (24, 25). Further, the presence of rifampin
resistance alone cannot be relied upon to predict INH resistance;
INH susceptibility ranges from ⱕ11% to ⬎40% of RIF-resistant
isolates, depending on the setting (28, 29). Thus, there remains a
need for rapid assessment of INH resistance in both research and
clinical care settings. It is important to note that molecular detec-
tion methods for INH resistance detect only a portion of muta-
tions leading to INH resistance (17), as reflected by the MTBDR
sensitivity in this series of 70.6% to 85.7%. The MTBDR-Plus
version 2.0 has improved INH detection, with sensitivities of 89%
to 100% in recent series (21, 22).
A second advantage of line probe assays is the ability to detect
nontuberculosis mycobacterium that are of particular clinical rel-
evance to the HIV-infected population, specifically MAC and M.
kansasii, and which may present similarly to or even in conjunc-
tion with TB infection. However, in this series, NTM were infre-
quently identified by either MGIT (2.2%) or Direct LPA (1.6%).
Direct LPA identified 7 NTM (MAC or M. kansasii) as well as 3
mixed infections with MAC/M. tuberculosis. Thus, detection of
NTMs may be of limited utility in a rapid M. tuberculosis diagnos-
tic in this study’s settings of particularly high TB prevalence but
may have increased utility in settings with higher prevalences of
MAC and/or M. kansasii (9, 30–32).
When considering line probe assay performance in compari-
son to culture-based methods, liquid-based mycobacterial culture
is limited by contamination rates that can be as high as 14.0% to
18.6% (7, 33, 34). The line probe invalid rates were substantially
lower, at 1.6% with MTBDR and 1.1% with Direct LPA, than the
MGIT contamination rate in this series of 4.2%.
The study used one MGIT culture as the comparator strategy
for M. tuberculosis identification, as access to culture-based M.
tuberculosis evaluation is often limited by cost and access in re-
source-limited settings, and a single MGIT has demonstrated a
higher yield for TB diagnosis than three solid-media M. tubercu-
losis cultures (35). Additional MGIT cultures may have identified
more culture-confirmed M. tuberculosis (35); however, the speci-
ficity of the LPAs was high, suggesting that substantial TB cases
were not being missed, and follow-up of available standard-of-
care testing identified only 7 (1.1%) additional M. tuberculosis
culture-positive cases. In addition, the study used three different
labs, which has the potential to introduce bias if laboratory tech-
niques differ across sites. To minimize this potential for bias, all
sites participated in external quality assurance proficiency testing
for AFB smear and mycobacterial culture, and personnel were
trained by Hain technicians on correct conduct of both LPAs.
The AFB⫹ rate in this series was high for an HIV-TB coinfected
population, in which AFB⫺ disease can be more frequent, partic-
ularly with advanced HIV. The median CD4⫹ cell count was 151/
mm3, indicating considerable immunosuppression; however,
only 18.2% of TB cases occurred in patients with CD4⫹ ⱕ50,
where the highest rates of AFB smear negative disease generally
occur (36). In addition, participants screened were suspected to
have high-risk TB, with either demonstrated AFB⫹ sputum or
1058 jcm.asm.org
empirical TB treatment planned, with 64.6% receiving TB treat-
ment within 30 days of enrollment. Thus, the population may
have been enriched for AFB⫹ specimens, given these eligibility
criteria. However, nearly 100 AFB smear-negative, MGIT M. tu-
berculosis-positive specimens were available for analysis of LPA
performance.
This study represents the largest series evaluating line probe
assays in a known HIV-infected population. Our findings sup-
ports the use of the MTBDR line probe assay for rapid M. tuber-
culosis drug resistance identification in HIV-infected TB suspects,
specifically in settings where rapid identification of INH resistance
is a priority. While AFB-negative sputum decreases test sensitivity,
use in this setting may still be considered, because MTBDR pro-
vided interpretable results in nearly half of smear-negative speci-
mens. The Direct LPA has improved sensitivity over MTBDR for
M. tuberculosis detection in AFB-negative specimens and per-
formed similarly to the Xpert MTB/RIF. However, the benefit of
identifying clinically relevant NTM in HIV-infected patients was
not clear in this series with a low prevalence of NTM. Use of line
probe assays that can detect NTM should be explored in other
settings of high HIV prevalence where increased NTM prevalence
may justify the additional testing.
ACKNOWLEDGMENTS
We thank the study participants, the site principal investigators and staff,
and the site laboratories for their exceptional efforts to conduct the study;
the data manager, Linda Wieclaw; the clinical trials specialist, Evelyn
Hogg; the field representative, Holly Boyd; the study’s laboratory technol-
ogist, David L. Shugarts; the laboratory data managers Courtney N.
Ashton and Anthony Bloom; and the community representative, Flavia
Miiro.
This work was supported by award number U01AI068636 from the
National Institute of Allergy and Infectious Diseases and the Statistical
and Data Management Center (SDMC) (UM1 AI068634) funded by the
National Institute of Allergy and Infectious Diseases. A.F.L. reports re-
search grant support to the University of California, San Francisco, from
Cepheid.
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